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Transcriptome and secretome analysis of mechanically stretched osteocytic cells reveals activation of signalling pathways implicated in bone regeneration and DNA-repair systems

ABSTRACT: Prescribed exercise has the potential to ameliorate tissue repair or regeneration and therefore is gaining increasing importance in regenerative rehabilitation. Osteocytes are an appealing type of bone cell to target by regenerative rehabilitation protocols due to their mechanosensitive and secretory nature. Making use of tandem liquid chromatography-mass spectrometry (LC-MSMS) as well as curated bioinformatics, we examined the secretome of mouse and human osteocytic bone cells cultured under cyclic tension delivered by a computer-controlled bioreactor. Quantitative SWATH MS analysis of the secretome secreted by mechanically stimulated cells revealed differential expression of 13 out of 759 secreted factors in mouse and 16 out of 276 in human osteocytic cells. GO enrichment analysis suggested that response to oxidative stress was the most predominant biological process in mouse and cholesterol or lipoprotein-related in human cells. Ossification and bone remodelling and manganese transport were 2 overrepresented process networks common to both mouse and human osteocytic cells

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Osteocyte

SUBMITTER: David Boocock

LAB HEAD: David John Boocock

PROVIDER: PXD020774 | Pride | 2021-06-08

REPOSITORIES: Pride

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Project description:BACKGROUND: Ischemia/reperfusion injury (IRI) is the leading cause of acute kidney injury (AKI). The current standard of care focuses on supporting kidney function, stating the need for more efficient and targeted therapies to enhance repair. Mesenchymal Stromal Cells (MSCs) and their secretome, either as conditioned medium (CM) or extracellular vesicles (EVs), have emerged as promising options for regenerative therapy, however, their full potential in treating AKI remains unknown.METHODS: In this study, we employed an in vitro model of chemically-induced ischemia using antimycin A combined with 2-deoxy-D-glucose to induce ischemic injury in proximal tubule epithelial cells. Afterwards, we evaluated the effects of MSC secretome, CM or EVs obtained from adipose tissue, bone marrow and umbilical cord, on ameliorating the detrimental effects of ischemia. To assess the damage and treatment outcomes, we analyzed cell morphology, mitochondrial health parameters (mitochondrial activity, ATP production, mass and membrane potential) and overall cell metabolism by metabolomics.RESULTS: Our findings show that ischemic injury caused cytoskeletal changes confirmed by disruption of the F-actin network, energetic imbalance as revealed by a 50% decrease in the oxygen consumption rate, increased oxidative stress, mitochondrial dysfunction and reduced cell metabolism. Upon treatment with MSC secretome, the morphological derangements were partly restored and ATP production increased by 40-50%, with umbilical cord-derived EVs being most effective. Furthermore, MSC treatment led to phenotype restoration as indicated by an increase in cell bioenergetics, including increased levels of glycolysis intermediates, as well as an accumulation of antioxidant metabolites.CONCLUSION: Our in vitro model effectively replicated the in vivo-like morphological and molecular changes observed during ischemic injury. Additionally, treatment with MSC secretome ameliorated proximal tubule damage, highlighting its potential as a viable therapeutic option for targeting AKI.

Project description:Acute respiratory distress syndrome (ARDS) is a catastrophic form of acute lung injury (ALI). The necessity for mechanical ventilation (MV) renders patients at risk for ventilator induced lung injury (VILI). Exposure to repetitive cyclic stretch (CS) and/or over-inflation exacerbates injury. Reducing tidal volume (VT) is the only therapeutic strategy shown to mitigate morbidity and mortality. Cyclic stretch has been shown to differentially regulate gene expression in part through the activation of mammalian mitogen-activated protein kinase (MAPK). Although these studies have shown both molecular and cellular alterations, no unifying hypothesis to explain MV-induced lung injury has emerged. In the current study, we hypothesized that coordinated expression of cyclic stretch (CS)-responsive genes relies on the presence of common CS-sensitive regulatory elements. To identify CS-responsive genes, we undertook a comparative examination of the gene expression profile of human bronchial epithelial airway (Beas-2B) cells in response to various injurious stimuli involved in the pathogenesis of acute lung injury (ALI)/Ventilator induced lung injury (VILI): cyclic stretch, tumor necrosis factor alpha (TNF-a), and lipopolysaccharide (LPS). Experiment Overall Design: Human Bronchial Epithelial Cells (Beas-B2) cells grown on silicon elastic plates coated with Type I collagen (Flexercell International, McKeesport, PA) were exposed to six regiments for 4 h: 1) control (static, [control]); 2) mechanical stretch (25 PKa, 30 cycles per min, [stretch]); 3) LPS (1 mcg/ml [LPS]); 4) TNF-Î± (20 ng/ml; [TNF]); 5) mechanical stretch plus LPS [LPS+S], and 6) mechanical stretch plus TNF-Î± [TNF+S]. Total RNA (duplicate experiments) was extracted using TRIZOL reagent (as per manufactures specifications) and purified using Qiagen mRNA purification Kit (as per manufacturers specifications). mRNA was hybridized to Affymetrix Human U133plus2.0 chips. Probe based analysis, background reduction, and quantile data normalization was performed in MeV 4.0 of TM4 using Robust Multi-array Average (RMA).

Project description:We previously reported that c-KIT+ human amniotic-fluid derived stem cells obtained from leftover samples of routine II trimester prenatal diagnosis (fetal hAFS) are endowed with regenerative paracrine potential driving pro-survival, pro-angiogenic, anti-fibrotic and proliferative effects on target cells. hAFS can be also isolated from III trimester clinical waste samples during C-section scheduled procedures (perinatal hAFS), thus offering an easily accessible alternative source. Nonetheless, little is known about their paracrine profile. Here we provide a detailed characterization of the hAFS total secretome (i.e. whole of the soluble paracrine factors released by the cells in their conditioned medium, hAFS-CM) and the extracellular vesicles (hAFS-EVs) within it as from II trimester fetal- versus III trimester perinatal cells. Fetal- and perinatal hAFS were characterized and subject to hypoxic preconditioning to enhance their paracrine potential. Their secretome formulations were analyzed in terms of protein and chemokine/cytokine content, with the EV cargo further investigated by RNA sequencing. While the appearance of fetal and perinatal hAFS, along with their corresponding secretome formulations was overlapping, the profiling of their paracrine cargo revealed some differences according to gestational stage and hypoxic preconditioning. While both cell sources provided paracrine formulations enriched with neurotrophic, immunomodulatory and endothelial stimulating factors, the more immature fetal hAFS secretome was defined by a more pronounced pro-angiogenic, pro-resolving and anti-aging profile over the perinatal one, while the corresponfing EV…. Moreover, small RNA profiling showed a relevant enrichment of microRNA in both the fetal and perinatal hAFS-EV cargo, with a steadily expressed pro-resolving miRNA core as reference molecular signature. Here we confirm that hAFS represents an appealing source of regenerative paracrine factors; the selection of either fetal or perinatal hAFS secretome formulations for future paracrine therapy should be evaluated considering the specific clinical scenario.

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Transcriptome and secretome analysis of mechanically stretched osteocytic cells reveals activation of signalling pathways implicated in bone regeneration and DNA-repair systems

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